Studies of the macromolecular structure of the nerve membrane involved in excitation were carried out by using the following four different methods. (1) A new electrophysiological method which combines the use of a real-time spectrum-analyzer with chemical nerve stimulants. (2) The method of radioactively labeling the proteins released from the interior of the squid giant axon under intracellular perfusion. (3) The method of radioactively labeling intact, excitable axons followed by SDS-acrylamide gel electrophoresis of axonal proteins. (4) The standard biochemical method of subcellular fractionation of nerve membrane homogenate. All four methods of investigation were found to be extremely useful in elucidation of the properties of proteins at excitable membrane sites.